modified-annexin-vpropidium-iodide-apoptosis-assay-for-accurate

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Primary kidney macrophage (PKM) cultures derived from goldfish hematopoietic tissues develop from early progenitors to mature macrophages in response to endogenous growth factor(s). When grown in vitro, PKM shift from a proliferative phase, where most of the proliferation and differentiation events take place, to a senescence phase, where there is cessation of proliferation and differentiation events and ultimately cell death through a process of apoptosis. The phenotypic changes of PKM from the proliferative to senescence phase are a reflection of specific changes in gene expression; therefore, comparison of gene expression patterns between the two phases should lead to the identification of macrophage genes directly involved in the positive and negative regulation of hematopoietic events, as well as genes that are modulated downstream from these regulatory points. Differential cross-screening of the proliferative phase PKM cDNA library using proliferative and senescence phase (32)P-labeled cDNA probes identified several differentially expressed genes. Specifically, initial screen of 9200 clones yielded 734 differential primary clones that were isolated and analyzed using a PCR-based secondary screen. The majority of these clone isolates encoded a single transcript as determined by PCR amplification of the primary clones. The secondary screen confirmed the differential expression of 306 clones (3.32% of the total number of screened clones). Two hundred and forty four clones were sequenced; 158 and 86 were preferentially expressed during proliferative and senescence phases, respectively. Several potential candidates of fish macrophage hematopoiesis were identified. These include, for example, zinc finger protein 147, nucleophosmin, 14-3-3 protein, adenine nucleotide translocator 2 (ANT2), granulin, survivin-1, and apoptosis inhibitor-5. In addition, several potential markers of macrophage differentiation and/or function were identified and their expression patterns characterized across three distinct stages of macrophage development in vitro. These include legumain, CD63, interferon-inducible protein, macrosialin (CD68), transcription factor MafB, and the molecular chaperone BiP/GRP78. These analyses will facilitate future characterization of macrophage developmental events by providing a more global perspective of various facets of macrophage hematopoiesis.

Differentially expressed genes that encode potential markers of goldfish macrophage development in vitro.

Differentiell exprimierte Gene, die potenzielle Marker von Goldfischen Makrophagen Entwicklung in vitro zu kodieren.

The colony-stimulating factors (CSFs) are a group of cytokines central to the hematopoiesis of blood cells, the modulation of their functional responses, as well as the maintenance of homeostasis and overall immune competence. This group consists of the macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF), and multi-CSF (IL-3). M-CSF and G-CSF are relatively lineage-specific, having a role in the proliferation, differentiation, and survival of macrophages, neutrophils, and their precursors. In contrast, GM-CSF and multi-CSF function at earlier stages of lineage commitment regulating the expansion and maturation of primitive hematopoietic progenitors. Colony stimulating factor production and degradation are strictly controlled, thus allowing for effective modulation of their biological functions in steady-state conditions as well as under periods of stress. Moreover, the mechanisms behind their expression and that of their cognate receptors ensures that their actions are tightly coordinated, within the context of a network of complex but finely tuned regulatory pathways derived from a variety of local and endocrine hematopoietic regulators. In this review we present some of the most salient information on CSF biology collected over the last three decades. We examine the gene and protein structure of each of the four CSFs and their corresponding receptors, and consider the main determinants behind their biological activities. The components responsible for their functional redundancy as well as the mechanisms that mediate their specificity are also discussed. Although most of available knowledge about CSFs is on human and mouse CSFs, an attempt was made to integrate recent findings in other systems in order to highlight a more widespread role for CSFs throughout evolution.

Regulation of myeloid development and function by colony stimulating factors.

Verordnung von myeloischen Entwicklung und Funktion von Kolonie-stimulierenden Faktoren.

Over 100 years after the first description of macrophages by Metchnikoff, there are still questions as to the mechanisms leading to the heterogeneity of their lineage. Current views are based on the mononuclear phagocyte system (MPS) theory, where all mammalian macrophages are derived from circulating blood monocytes and ultimately from hematopoietic stem cells in the bone marrow. Our studies on the regulation of fish macrophage development, suggested that teleosts have alternate pathways of monopoiesis, which undoubtedly contribute to macrophage heterogeneity in the goldfish. Macrophage heterogeneity has been attributed to a network of positive and negative regulators of macrophage development, including soluble mediators known as colony-stimulating factors of which two (M-CSF and GM-CSF) promote formation and growth of mature macrophages. In contrast to our knowledge of CSFs and their receptors in mammals, there is no published information about fish macrophage CSFs. Since fish macrophages generate their own growth factors, it is reasonable to assume that pathways of fish macrophage development and hematopoiesis may be distinct from those of mammalian macrophages. More importantly, the presence of fish progenitor/stem cells and developing macrophages in long-term cultures, allowed us to address pathways of macrophage differentiation, which could not be addressed in mammalian macrophage culture systems. Characterization of primary kidney macrophage (PKM) cultures from goldfish hematopoietic tissues (kidney) indicated that three distinct subpopulations developed in response to endogenous macrophage growth factors. These macrophage subpopulations expressed several differentiation markers, including the hematopoietic stem cell antigen AC133, c-kit, granulin, CD63, macrosialin, c/EBPbeta, legumain, and the colony-stimulating factor receptor-1 (CSF-1R). In the goldfish, there appeared to be a stringent control between those early progenitors that self-renewed, and those that were recruited into the maturation pathways. We report that upon commitment, goldfish macrophages developed through two distinct differentiation pathways: one consistent with the "classical" pathway (MPS) of macrophage development (progenitors-->monocytes-->mature macrophages), and an "alternate" pathway (AP-macrophages) where mature macrophages appeared to rapidly develop from early progenitors in the absence of an intermediate monocyte stage. AP-macrophages represent a unique subset of spontaneously growing cells. Their self-renewal was promoted by endogenous macrophage growth factors (MGF), and effectively controlled by a novel soluble form of the CSF-1R (sCSF-1R). The discovery of sCSF-1R in the goldfish highlights the inherent complexity in the hematopoietic regulatory machinery of teleosts.

Development of goldfish macrophages in vitro.

Entwicklung von Goldfischen Makrophagen in vitro.

Macrophage colony-stimulating factor (CSF-1) is the principal regulator of the survival, proliferation, and differentiation of macrophages and their precursors. CSF-1 activity is tightly controlled through mechanisms regulating gene expression of CSF-1 and its membrane-bound receptor (CSF-1R), as well as by receptor-mediated endocytosis, metabolic processing, and inhibition of downstream signaling. Herein we describe a novel mechanism for control of CSF-1 activity. Spontaneously growing goldfish (Carassius auratus L.) macrophages actively produced a soluble form of CSF-1R (sCSF-1R) that appears to compete for ligand binding with membrane CSF-1R. The sCSF-1R transcript encodes only for the ligand-binding portion of the receptor, but is derived from a full-length mRNA species as determined by sequence analysis. Gene expression was associated with decreased proliferation and differentiation of primary macrophages, decreased growth factor activity in culture supernatants, and marked phenotypic changes that culminated in apoptotic cell death. Recombinant sCSF-1R inhibited macrophage proliferation at nanomolar concentrations. Antibodies against recombinant sCSF-1R identified native sCSF-1R in primary macrophage culture supernatants and fish serum, and suggested the presence of endogenous mechanisms temporally regulating sCSF-1R release. This is the first report of a soluble CSF-1R and points to intrinsic mechanisms of hematopoietic control that may be conserved across evolution in discrete self-renewing macrophage populations.

A novel soluble form of the CSF-1 receptor inhibits proliferation of self-renewing macrophages of goldfish (Carassius auratus L.).

Eine neuartige l&#xF6;sliche Form des CSF-1-Rezeptor hemmt die Proliferation von selbst erneuernden Makrophagen von Goldfisch (Carassius auratus L.).

Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike mammalian granulins, which possess 7.5 repeats of a characteristic signature of 12 cysteine residues, the goldfish granulin encoded a putative peptide possessing only 1.5 cysteine repeats. Northern blot and real-time PCR analyses indicated that goldfish granulin was expressed only in the hematopoietic tissues of the goldfish, specifically the kidney and spleen, and in activated peripheral blood mononuclear cells. We expressed granulin using a prokaryotic expression system and produced an affinity-purified rabbit anti-goldfish granulin IgG. Recombinant goldfish granulin induced a dose-dependent proliferative response of goldfish macrophages that was inversely related to the myeloid differentiation stage of the cells studied. The highest proliferative response was observed in macrophage progenitor cells and monocytes. This proliferative response of macrophages was abrogated by the addition of anti-granulin IgG. These results indicate that goldfish granulin is a growth factor that positively modulates cell proliferation at distinct junctures of macrophage differentiation.

A novel hematopoietic granulin induces proliferation of goldfish (Carassius auratus L.) macrophages.

Granulins sind eine Gruppe von stark konservierten Wachstumsfaktoren, die aus einer Vielzahl von Organismen, die &uuml;ber die Metazoa beschrieben wurden. In dieser Studie Goldfisch H&uuml;lle war einer der am h&auml;ufigsten festgestellte Transkripte in das differenzielle Kreuz-Screening von Makrophagen cDNA-Bibliotheken und wurde bevorzugt in proliferierenden Makrophagen ausgedr&uuml;ckt. Im Gegensatz zu S&auml;ugetieren Granulins, die welche 7,5 Wiederholungen einer charakteristischen Signatur 12 Cystein-R&uuml;ckst&auml;nde zu besitzen, codiert die Goldfisch-H&uuml;lle ein mutma&szlig;liches Peptid besitzen nur 1,5 Cystein-Wiederholungen. Nordfleck und Echtzeit-PCR-Analysen angegeben, dass Goldfische-H&uuml;lle wurde nur in den h&auml;matopoetischen Geweben von Goldfisch, insbesondere der Niere und Milz, ausgedr&uuml;ckt und in aktiviert peripheren mononukle&auml;ren Blutzellen. Wir H&uuml;lle mit einer prokaryotischen Expressionssystem ausgedr&uuml;ckt und produziert eine Affinit&auml;t gereinigt Kaninchen anti-goldfish H&uuml;lle IgG. Rekombinante Goldfisch H&uuml;lle induziert eine dosisabh&auml;ngige proliferative Antwort der Goldfisch Makrophagen, die umgekehrt auf die B&uuml;hne myeloische Differenzierung der Zellen studiert verwandt war. Die h&ouml;chste proliferative Reaktion wurde in Vorl&auml;uferzellen von Makrophagen und Monozyten beobachtet. Diese proliferative Reaktion der Makrophagen wurde durch die Zugabe von IgG Anti-H&uuml;lle aufgehoben. Diese Ergebnisse zeigen, dass die Goldfisch-H&uuml;lle einen Wachstumsfaktor ist, der Zellproliferation an unterschiedlichen Stellen der Differenzierung von Makrophagen positiv moduliert.

Eine neuartige h&auml;matopoetischen H&uuml;lle induziert Vermehrung der Goldfische (Carassius Auratus L.) Makrophagen.

Fc gamma receptors (FcgammaRs) contribute to the internalization of large and small immune complexes through phagocytosis and endocytosis, respectively. The molecular processes underlying these internalization mechanisms differ dramatically and have distinct outcomes in immune clearance and modulation of cell function. However, it is unclear how the same receptors (FcgammaR) binding to identical ligands (IgG) can elicit such distinct responses. We and others have shown that Syk kinase, Src-related tyrosine kinases (SRTKs) and phosphatidyl inositol 3-kinases (PI3K) play important roles in FcgammaR phagocytosis. Herein, we demonstrate that these kinases are not required for FcgammaR endocytosis. Endocytosis of heat-aggregated IgG (HA-IgG) by COS-1 cells stably transfected with FcgammaRIIA or chimeric FcgammaRI-gamma-gamma (EC-TM-CYT) was not significantly altered by PP2, piceatannol, or wortmannin. In contrast, phagocytosis of large opsonized particles (IgG-sensitized sheep erythrocytes, EA) was markedly reduced by these inhibitors. These results were confirmed in primary mouse bone marrow-derived macrophages and freshly isolated human monocytes. Levels of receptor phosphorylation were similar when FcgammaRIIA was cross-linked using HA-IgG or EA. However, inhibition of FcgammaR phosphorylation prevented only FcgammaR phagocytosis. Finally, biochemical analyses of PI3K(p85)-Syk binding indicated that direct interactions between native Syk and PI3K proteins are differentially regulated during FcgammaR phagocytosis and endocytosis. Overall, our results indicate that FcgammaR endocytosis and phagocytosis differ dramatically in their requirement for Syk, SRTKs, and PI3K, pointing to striking differences in their signal transduction mechanisms. We propose a competitive inhibition-based model in which PI3K and c-Cbl play contrasting roles in the induction of phagocytosis or endocytosis signaling cascades.

Differential kinase requirements in human and mouse Fc-gamma receptor phagocytosis and endocytosis.

FC-Gamma-Rezeptoren (FcgammaRs) tragen zur Internalisierung von gro&szlig;en und kleinen Immunkomplexe durch Phagozytose und Endozytose, beziehungsweise. Die molekularen Prozessen zugrunde liegen, diese Internalisierung Mechanismen unterscheiden sich dramatisch und haben unterschiedliche Ergebnisse im Immunsystem Freiraum und Modulation der Zellfunktion. Aber es ist unklar, wie die gleichen Rezeptoren (FcgammaR) Bindung an identische Liganden (IgG) kann solche unterschiedlichen Reaktionen hervorrufen. Wir und andere haben diese Kinase Syk Src-bezogene Tyrosinkinasen (SRTKs) und Phosphatidyl-Inositol-3-Kinasen (PI3K) eine wichtige Rolle in FcgammaR Phagozytose spielen gezeigt. Hier zeigen wir, dass diese Kinasen nicht f&uuml;r FcgammaR Endozytose erforderlich sind. Endozytose von Hitze aggregierte IgG (HA-IgG) von COS-1 Zellen die stabil mit FcgammaRIIA transfected oder Chim&auml;ren FcgammaRI-Gamma-Gamma (EG-TM-CYT) wurde durch PP2, Piceatannol oder Wortmannin nicht wesentlich ver&auml;ndert. Im Gegensatz dazu opsonized Phagozytose von gro&szlig;en Partikeln (IgG sensibilisiert Schaf-Erythrozyten, EA) wurde durch diese Inhibitoren deutlich reduziert. Diese Ergebnisse wurden prim&auml;re Maustaste Knochenmark abgeleiteten Makrophagen und frisch isolierten menschlichen Monozyten best&auml;tigt. Ebenen der Rezeptor Phosphorylierung &auml;hnelten sich, wenn FcgammaRIIA mit HA-IgG oder EA vernetzt war. Allerdings verhinderten die Hemmung der FcgammaR Phosphorylierung nur FcgammaR Phagozytose. Schlie&szlig;lich biochemische Analysen der PI3K (p85)-Syk Bindung angegeben, dass direkte Interaktionen zwischen systemeigenen Syk und PI3K Proteine differentiell w&auml;hrend FcgammaR Phagozytose und Endozytose reguliert werden. Insgesamt zeigen unsere Ergebnisse, dass FcgammaR Endozytose und Phagozytose drastisch in ihrer Anforderung f&uuml;r Syk, SRTKs und PI3K, auf markante Unterschiede in der Signal-Transduktion-Mechanismen unterscheiden. Wir bieten ein wettbewerbsf&auml;higes Hemmung basierende Modell in der PI3K und c-Cbl spielen gegens&auml;tzlichen Rollen in die Induktion von Phagozytose oder Endozytose Signalisierung Kaskaden.

Differenzielle Kinase Anforderungen in Mensch und Maus Fc-Gamma-Rezeptor Phagozytose und Endozytose.

The present paradigm dictates that phagocytosis is accomplished mainly by 'professional' phagocytes (such as macrophages and monocytes), whereas B cells lack phagocytic capabilities. Here we demonstrate that B cells from teleost fish have potent in vitro and in vivo phagocytic activities. Particle uptake by B cells induced activation of 'downstream' degradative pathways, leading to 'phagolysosome' formation and intracellular killing of ingested microbes. Those results indicate a previously unknown function for B cells in the innate immunity of these primitive animals. A considerable proportion of Xenopus laevis B cells were also phagocytic. Our findings support the idea that B cells evolved from an ancestral phagocytic cell type and provide an evolutionary framework for understanding the close relationship between mammalian B lymphocytes and macrophages.

B lymphocytes from early vertebrates have potent phagocytic and microbicidal abilities.

Das gegenw&auml;rtige Paradigma diktiert, dass die Phagozytose haupts&auml;chlich durch &quot;professionelle&quot; Fresszellen (z. B. Makrophagen und Monozyten), vollendet wird, w&auml;hrend die B-Zellen phagozytische Funktionen fehlen. Hier zeigen wir, dass B-Zellen von teleost Fische potente in vitro und in vivo phagozytische T&auml;tigkeit haben. Partikel Aufnahme durch B-Zellen induziert Aktivierung von &#39;downstream&#39; Degradations &Uuml;bertragungswege bei &#39;Phagolysosome&#39; Bildung und intrazellul&auml;re T&ouml;tung von aufgenommenen Mikroben. Diese Ergebnisse zeigen eine bisher unbekannte Funktion f&uuml;r B-Zellen in der angeborenen Immunit&auml;t dieser primitive Tiere. Ein erheblicher Anteil der Xenopus Laevis B-Zellen waren auch phagozytische. Unsere Erkenntnisse unterst&uuml;tzen die Idee, dass B-Zellen entwickelte sich aus einer uralten phagozytische Zelle geben und einen evolution&auml;ren Rahmen zum Verst&auml;ndnis der engen Beziehung zwischen S&auml;ugetier-B-Lymphozyten und Makrophagen bilden.

B-Lymphozyten aus fr&uuml;hen Wirbeltieren haben starke phagozytische und Mikrobizide F&auml;higkeiten.

The integral membrane protein known as prominin was first identified on the apical surface of mouse neural epithelial cells as well as on the surface of human haematopoietic progenitor cells. This report describes a prominin-like sequence and expression analysis of the prominin in the goldfish. The predicted amino acid sequence for goldfish prominin shares all of the hallmark structural characteristics of the prominin family, however the relatedness assessed using the percent amino acid identity indicated that goldfish prominin cannot be placed into the current mammalian dichotomy of type 1 or 2. The real time PCR analyses indicated that prominin was broadly expressed in different tissues with particularly high levels observed in the kidney and gill of the goldfish. Goldfish prominin was also found to be differentially expressed in subpopulations of in vitro-derived goldfish macrophages, with the highest expression observed in progenitor cells.

Cloning and expression analysis of goldfish (Carassius auratus L.) prominin.

Das integrale Membranprotein, bekannt als Prominin wurde zun&auml;chst auf die apikale Oberfl&auml;che der Maus neuronale Epithelzellen sowie auf der Oberfl&auml;che des menschlichen h&auml;matopoetische Vorl&auml;uferzellen identifiziert. Dieser Bericht beschreibt eine Prominin-&auml;hnlichen Sequenz und Ausdruck-Analyse der Prominin in der Goldfisch. Der vorhergesagten Aminos&auml;uresequenz f&uuml;r Goldfische Prominin teilt alle strukturellen Merkmale der Familie Prominin, Markenzeichen, aber der Verwandtschaft, die anhand der prozentualen Aminos&auml;ure-Identit&auml;t lie&szlig;en erkennen, dass diese Goldfische Prominin in die aktuelle S&auml;ugetier-Dichotomie des Typs 1 oder 2 platziert werden kann nicht. Die Real-Time-PCR-Analysen erkennen, dass diese Prominin weitgehend in verschiedenen Geweben mit einem besonders hohen beobachtet in der Niere und Gill die Goldfish ausgedr&uuml;ckt wurde. Ferner ergab die Goldfisch Prominin differentiell werden in Subpopulationen von in Vitro-abgeleitete Goldfisch Makrophagen, mit dem h&ouml;chsten Ausdruck beobachtet in Vorl&auml;uferzellen ausgedr&uuml;ckt.

Klonierung und Expression Analyse der Goldfisch (Carassius Auratus L.) Prominin.

Der angeborenen Immunantwort des fr&uuml;hen Wirbeltiere, wie z. B. Knochenfische, spielen eine zentrale Rolle in der Host-Abwehr gegen Infektionskrankheiten und eine der wichtigsten Effektorzellen der angeborenen Immunit&auml;t sind Makrophagen. In Reihenfolge f&uuml;r Makrophagen Host Verteidigung erwiesen, dass sie alle Zeiten in den Geweben ihres Wirtes und allem anwesend sein m&uuml;ssen muss der Host rasant Makrophagen Zahlen in Zeiten der Not k&ouml;nnen. Blutbildung ist ein Prozess der Bildung und Entwicklung von reifer Blutzellen, einschlie&szlig;lich Makrophagen. Blutbildung wird gesteuert durch l&ouml;sliche Faktoren, sogenannte Zytokine, die Ver&auml;nderungen der Transkriptionsfaktoren in den Zielzellen, die Zelle Schicksal &Auml;nderungen und die endg&uuml;ltige Entwicklung der spezifischen Effektorzellen beeinflussen. Prozesse in Makrophagen Entwicklung haben weitgehend von S&auml;ugetier-Modellorganismen abgeleitet wurde. Jedoch wurden neue Zuf&uuml;hrungen zum Verst&auml;ndnis der Entwicklung der Makrophagen in Knochenfische, eine Gruppe von Organismen vorgenommen, die stark auf ihre angeborene Immunabwehr. Unser Verst&auml;ndnis von den Wachstumsfaktoren beteiligt teleost Makrophage-Entwicklung, als auch die Rezeptoren und regulatorische Mechanismen zu kontrollieren hat betr&auml;chtlich zugenommen. Dar&uuml;ber hinaus entstanden Modellorganismen wie dem Zebrafisch als wichtige Instrumente zur F&ouml;rderung unser Verst&auml;ndnis der transkriptionelle Kontrolle der Zelle Entwicklung bei Fischen ebenso wie bei S&auml;ugetieren. Diesen Bericht unterstreicht die neuen Zuf&uuml;hrungen in unserem Verst&auml;ndnis der teleost Makrophage-Entwicklung. Konzentrierten wir uns auf den Wachstumsfaktoren identifiziert, um bei der Regulierung der Makrophagen Entwicklung aus einer Zelle Vorl&auml;uferzellen in einer funktionalen Makrophagen wichtig sein und diskutieren die wichtige Transkriptionsfaktoren, die festgestellt wurden Funktion in teleost Blutbildung. Wir beschreiben auch die Ergebnisse der in-vivo-Untersuchungen, die Beobachtungen, die in-vitro verst&auml;rkt haben und haben wesentlich verbessert, die Relevanz und die Bedeutung der Verwendung von teleost Fische als Modellorganismen f&uuml;r Entwicklungsprozesse zu studieren.

Entwicklung von Makrophagen Friedfischrevieren Fische.

The innate immune responses of early vertebrates, such as bony fishes, play a central role in host defence against infectious diseases and one of the most important effector cells of innate immunity are macrophages. In order for macrophages to be effective in host defence they must be present at all times in the tissues of their host and importantly, the host must be capable of rapidly increasing macrophage numbers during times of need. Hematopoiesis is a process of formation and development of mature blood cells, including macrophages. Hematopoiesis is controlled by soluble factors known as cytokines, that influence changes in transcription factors within the target cells, resulting in cell fate changes and the final development of specific effector cells. The processes involved in macrophage development have been largely derived from mammalian model organisms. However, recent advancements have been made in the understanding of macrophage development in bony fish, a group of organisms that rely heavily on their innate immune defences. Our understanding of the growth factors involved in teleost macrophage development, as well as the receptors and regulatory mechanisms in place to control them has increased substantially. Furthermore, model organisms such as the zebrafish have emerged as important instruments in furthering our understanding of the transcriptional control of cell development in fish as well as in mammals. This review highlights the recent advancements in our understanding of teleost macrophage development. We focused on the growth factors identified to be important in the regulation of macrophage development from a progenitor cell into a functional macrophage and discuss the important transcription factors that have been identified to function in teleost hematopoiesis. We also describe the findings of in vivo studies that have reinforced observations made in vitro and have greatly improved the relevance and importance of using teleost fish as model organisms for studying developmental processes.

Development of macrophages of cyprinid fish.

The most likely route of entry of infection following oral exposure to transmissible spongiform encephalopathies (TSE) is via the immunologically active Peyer's patches (PP). These secondary lymphoid organs appear to be the potential route for prion neuroinvasion. However, the molecular mechanisms involved in the uptake of the infectious prion agent and progression of disease remain still unclear. This investigation examined the changes in gene expression in PP following oral exposure of cattle to bovine spongiform encephalopathy (BSE) agents. The gene expression patterns in PP from cows 12 mo after BSE challenge were compared with controls using a microarray platform containing 24,000 oligonucleotides representing 16,846 unique gene loci and 5943 Expressed Sequence Tag (EST) from bovine genome. Between the challanged and control animals, 90 genes and 16 EST were identified as significantly differentially, expressed (>2.0-fold change): 36 were upregulated and 70 were downregulated. Of these genes, five were found to be related to immune function. Major histocompatibility complex (MHC) class II, MHC class II DQ alpha, L-RAP, and two hypothetical proteins. Differentially expressed genes related to cellular and metabolic processes including development and maturation of cells in the PP were also identified. In this context, the potential impacts of these gene expression changes in PP on BSE development are discussed.

Microarray analysis of differentially expressed genes from Peyer's patches of cattle orally challenged with bovine spongiform encephalopathy.

Der wahrscheinlichste Weg des Eintrags der Infektion nach oraler Exposition gegen&uuml;ber transmissiblen spongiformen Enzephalopathien (TSE) ist &uuml;ber die immunologisch aktive Peyer Flecken (PP). Diese sekund&auml;ren lymphatischen Organe scheinen die m&ouml;gliche Route f&uuml;r Prion-Neuroinvasion. Die molekularen Mechanismen, die die Aufnahme von der infekti&ouml;sen Prionen-Agent und Fortschreiten der Krankheit beteiligt bleiben jedoch noch unklar. Dieser Untersuchung untersucht die Ver&auml;nderungen in der Genexpression in PP nach oraler Exposition von Rindern bovine spongiforme Enzephalopathie (BSE) Arbeitsstoffe. Die Genausdruck Muster in PP von K&uuml;hen 12 Mo nach BSE-Challenge wurden mit Steuerelementen mithilfe einer Microarray-Plattform mit 24.000 Oligonukleotide, 16.846 einzigartige gen-Loci und 5943 &auml;u&szlig;erte Sequenz Tag (EST) aus Rindern Genom verglichen. Zwischen der Tiere Verkleidungswettbewerb und, 90 Gene und 16 EST wurden identifiziert, wie signifikant differentiell ausgedr&uuml;ckt (&gt; 2.0-fold &Auml;nderung): 36 waren herraufreguliert und 70 Downregulated. Dieser Gene fanden f&uuml;nf Immunfunktion abstammte. Major Histocompatibility complex (MHC)-Klasse II MHC Klasse II-DQ Alpha, L-RAP und zwei hypothetische Proteine. Differentiell exprimierten Gene im Zusammenhang mit Mobilfunk und metabolische Prozesse, einschlie&szlig;lich der Entwicklung und Reifung von Zellen in der PP wurden auch identifiziert. In diesem Zusammenhang werden die m&ouml;glichen Auswirkungen dieser Gen-Ausdruck-&Auml;nderungen in PP auf BSE Entwicklung diskutiert.

Microarray Analyse differentiell exprimierten Genen von Peyer die Patches von Vieh m&uuml;ndlich mit der bovinen spongiformen Enzephalopathie in Frage gestellt.

The advent of flow cytometry-based applications has significantly impacted the study of cellular apoptosis. Propidium iodide (PI) is a commonly used viability stain in these studies. Unfortunately, we find that conventional Annexin V/PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment. Both primary cells and cell lines are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence. This distribution spans a wide range of animal models including mice, swine, avian, and teleost fish and potentially affects up to 1016 out of 1019 of peer-reviewed papers published in this area since 1995. We show that the primary ramifications from these findings relate to cells experiencing changes in RNA content. Virally infected cells, for example, are qualified as undergoing apoptosis in response to infection based on conventional staining protocols; in fact, these cells are alive and actively producing viral RNA that can serve to produce additional infectious viral particles. Based on our observations we propose a modified protocol, show that it overcomes previous drawbacks for this technique, and that it will allow for more accurate assessment of cell death across various platforms.

Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.

Das Aufkommen der Flow Cytometry-basierten Anwendungen hat das Studium der zellul&auml;ren Apoptose erheblich beeinflusst. Propidium Jodid (PI) ist eine h&auml;ufig verwendete Lebensf&auml;higkeit Fleck in diesen Studien. Leider finden wir, dass konventionelle Annexin V/PI-Protokolle zu eine betr&auml;chtliche Anzahl von falsch positiven Ereignisse f&uuml;hren (bis zu 40 %), die PI Beflecken RNS innerhalb des zytoplasmatischen Fachs zugeordnet sind. Prim&auml;rzellen und Zelllinien betroffen sind, mit gro&szlig;en Zellen (nukleare: zytoplasmatische Verh&auml;ltnisse &lt; 0,5) zeigen das h&ouml;chste vorkommen. Diese Distribution umfasst eine Vielzahl von Tiermodellen, darunter M&auml;use, Schweinepest, Vogelgrippe, und teleost Fische und potenziell betrifft bis zu 1016 aus 1019 Peer-Review-Papiere, die in diesem Bereich seit 1995 ver&ouml;ffentlicht. Wir zeigen, dass die prim&auml;ren Konsequenzen aus diesen Erkenntnissen Zellen erleben RNA Content-&Auml;nderungen betreffen. Viral infizierte Zellen, sind beispielsweise qualifiziert als Apoptose in Reaktion auf eine Infektion, die auf der Grundlage konventioneller F&auml;rbung Protokolle unterzogen; Diese Zellen sind in der Tat lebendig und aktiv produzierenden viraler RNA, die dienen kann, um zus&auml;tzliche ansteckende virale Partikel zu produzieren. Aufgrund unserer Beobachtungen, dass schlagen wir eine ge&auml;nderte Protokoll, zeigen Sie, dass es fr&uuml;here Nachteile dieser Technik &uuml;berwindet und es f&uuml;r eine genauere Beurteilung des Zelle Todes &uuml;ber verschiedene Plattformen hinweg erm&ouml;glicht.

Konventionelle Apoptose-Assays, die mit Propidium Jodid generieren eine erhebliche Anzahl der Fehlalarme, die genaue Bewertung des Zelle Todes zu verhindern.

Erratum zu &quot;Klonen und Ausdruck Analyse der Goldfische (Carasius Auratus L.) Prominin&quot; [Fische Schalentiere Immunol. 22 (2007) 308-317].

Erratum to "Cloning and expression analysis of goldfish (Carasius auratus L.) prominin" [Fish Shellfish Immunol. 22 (2007) 308-317].

Phagocytosis provides a critical first line of defense against invading pathogens. Engagement of particles through receptor-mediated binding precedes internalization and induction of cellular antimicrobial responses. Phagocytes have the capacity to differentially regulate binding and internalization processes through changes in their receptor profile and modulation of downstream events. This is necessary for the intricate control of phagocytic antimicrobial responses. Several methods are available for evaluation of phagocytosis. Unfortunately, none allow for accurate quantitation of both binding and internalization events. To overcome these limitations, we have developed a novel phagocytosis assay based on a multi-spectral imaging flow cytometry platform. This assay discriminates between internalized and surface-bound particles in a statistically robust manner and allows multi-parametric analysis of phagocytosis and downstream anti-microbial responses. We also devised a novel approach for examination of phagolysosome fusion, which provides an improved capacity for quantitative assessment of phagolysosome fusion in mixed populations of intact cells. Importantly, our approaches are likely amenable to a broad range of comparative model systems based on our examination of murine RAW 264.7 cells and a goldfish primary kidney macrophage (PKM) model system. The latter allowed us to examine the evolutionary conservation of phagocytic antimicrobial responses in a lower vertebrate model. While it has been previously reported that mixed populations of these macrophage cultures are phagocytic, it remained unclear if sub-populations within them contributed differentially to this activity. In accordance with higher vertebrate models, we found that differentiation along the macrophage pathway leads to an increased capacity for phagocytosis in goldfish PKM. Interestingly, cellular activation differentially regulated particle internalization in PKM monocyte and mature macrophage subsets. We also found differential regulation of phagolysosome fusion and downstream production of reactive oxygen intermediates (ROI). The temporal activation of specific phagocytic antimicrobial responses at distinct stages of PKM differentiation suggests specialization within the macrophage compartment early in evolution, geared to meet specific host immunity requirements within specialized niches.

Macrophage activation differentially modulates particle binding, phagocytosis and downstream antimicrobial mechanisms.

Phagozytose stellt einen kritischen ersten Linie der Verteidigung gegen eindringende Krankheitserreger. Engagement von Partikeln durch Rezeptor-vermittelte Bindung vorausgeht Internalisierung und Induktion der zellul&auml;ren antimikrobielle Antworten. Fresszellen haben die F&auml;higkeit zur Bindung und Internalisierung Prozesse durch &Auml;nderungen in ihrem Rezeptor-Profil und Modulation der nachgeschalteten Ereignisse differentiell zu regulieren. Dies ist notwendig f&uuml;r die komplizierte Kontrolle der phagozytische antimikrobielle Antworten. F&uuml;r die Bewertung der Phagozytose stehen mehrere Methoden zur Auswahl. Leider zulassen nichts f&uuml;r genaue quantitative Bestimmung der Bindung und Internalisierung Veranstaltungen. Diese Einschr&auml;nkungen zu &uuml;berwinden, haben wir eine neuartige Phagozytose-Assay basiert auf einer multispektralen bildgebenden flie&szlig;en Cytometry-Plattform entwickelt. Diese Probe zwischen verinnerlichten und Oberfl&auml;che gebundene Teilchen in einer statistisch zuverl&auml;ssige Weise diskriminiert und Multiparameter-Analyse der Phagozytose und nachgeschaltete Anti-mikrobiellen Reaktionen erm&ouml;glicht. Wir entwickelten auch einen neuen Ansatz zur Untersuchung von Phagolysosome Fusion, bietet eine verbesserte Kapazit&auml;t f&uuml;r quantitative Beurteilung der Phagolysosome Fusion in gemischten Populationen von intakten Zellen. Wichtig ist, d&uuml;rften unsere Ans&auml;tze geneigt sein, eine breite Palette von vergleichenden Modellsysteme basiert auf unsere Pr&uuml;fung des murinen RAW 264.7 Zellen und Makrophagen (PKM) Modellsystem Goldfisch prim&auml;ren Nieren. Letzteres konnten wir die evolution&auml;re Erhaltung der phagozytische antimikrobielle Antworten in einem niedrigeren Vertebraten Modell zu untersuchen. W&auml;hrend zuvor berichtet wurde, dass gemischte Bev&ouml;lkerung dieser Makrophagen-Kulturen phagozytische sind, blieb unklar, ob Subpopulationen innerhalb dieser differentiell zu dieser T&auml;tigkeit beigetragen. Entsprechend h&ouml;here vertebrate Modelle fanden wir, dass eine erh&ouml;hte F&auml;higkeit zur Phagozytose in Goldfisch PKM Differenzierung entlang der Makrophage-Weg f&uuml;hrt. Interessanterweise reguliert zellul&auml;re Aktivierung differentiell Partikel Internalisierung in PKM Monozyte und Reifen Makrophagen Teilmengen. Wir fanden auch differenzielle Verordnung von Phagolysosome Fusion und nachgelagerte Produktion von reaktiven Sauerstoff Zwischenprodukte (ROI). Die zeitliche Aktivierung von bestimmten phagozytische antimikrobielle Antworten auf unterschiedlichen Stufen der PKM Differenzierung schl&auml;gt Spezialisierung innerhalb der Makrophagen-Fach fr&uuml;h in der Evolution, an bestimmten Host Immunit&auml;t Anforderungen in speziellen Nischen ausgerichtet.

Makrophagen Aktivierung moduliert differentiell Partikel binden, Phagozytose und nachgeschaltete antimikrobielle Mechanismen.

Early activation and coordination of innate defenses are critical for effective responses against infiltrating pathogens. Rapid engagement of immune cells provides a critical first line of defense soon after pathogen infiltration. Activation leads to a well-orchestrated set of events that sees the induction and regulation of intracellular and extracellular antimicrobial defenses. An array of regulatory mediators, highly toxic soluble molecules, degradative enzymes and antimicrobial peptides provides maximal protection against a wide range of pathogens while limiting endogenous damage to host tissues. In this review we highlight recent advances in our understanding of innate cellular antimicrobial responses of teleost fish and discuss their implications to cell survival, immunomodulation and death. The evolutionary conservation of these responses is a testament to their effectiveness against pathogen infiltration and their commitment to effective maintenance of host homeostasis. Importantly, recent developments in teleost fish systems have identified novel host defense strategies that may be unique to this lower vertebrate group or may point to previously unknown innate mechanisms that also play a significant role in higher vertebrate host immunity.

Antimicrobial mechanisms of fish leukocytes.

Fr&uuml;he Aktivierung und Koordinierung der angeborenen Abwehr sind wichtig f&uuml;r wirksame Ma&szlig;nahmen gegen Krankheitserreger zu infiltrieren. Schnelle Engagement von Immunzellen bietet einen kritischen ersten Linie der Verteidigung bald nach Erreger eindringen. Aktivierung f&uuml;hrt ein instrumentiertes Satz von Ereignissen, der die Induktion sieht und Regulierung der intrazellul&auml;ren und extrazellul&auml;ren antimikrobielle Abwehr. Ein Array von regulatorischen Mediatoren, hochgiftige l&ouml;sliche Molek&uuml;le, Degradations Enzyme und antimikrobielle Peptide bietet maximalen Schutz gegen ein breites Spektrum von Erregern w&auml;hrend den endogenen Schaden auf Host-Gewebe. In diesem Bericht wir markieren die j&uuml;ngsten Fortschritte im Verst&auml;ndnis der angeborenen zellul&auml;re antimikrobielle Antworten teleost Fische und ihre Implikationen f&uuml;r Zelle &uuml;berleben, Immunmodulation und Tod zu diskutieren. Die evolution&auml;re Erhaltung dieser Antworten ist ein Beweis f&uuml;r ihre Wirksamkeit gegen Erreger eindringen und ihr Engagement f&uuml;r eine effektive Wartung der Host-Hom&ouml;ostase. Wichtig ist, haben j&uuml;ngste Entwicklungen in teleost Fische Systeme Roman Host Verteidigungsstrategien identifiziert, die m&ouml;glicherweise f&uuml;r diese niedrigeren Vertebraten Gruppe eindeutig oder verweist m&ouml;glicherweise auf bisher unbekannte angeborene Mechanismen, die auch eine bedeutende Rolle in h&ouml;heren Wirbeltieren Host Immunit&auml;t zu spielen.

Antimikrobielle Mechanismen der Fisch Leukozyten.

Breaking the long-held paradigm that primary B cells are not phagocytic, several studies have demonstrated recently that B cells from fish, amphibians, and reptilians have a significant phagocytic capacity. Whether such capacity has remained conserved in certain mammalian B cell subsets is presently an enigma. Here, we report a previously unrecognized ability of PerC B-1a and B-1b lymphocytes to phagocytose latex beads and bacteria. In contrast, B-2 lymphocytes had an almost negligible ability to internalize these particles. Upon phagocytosis, B-1a and B-1b cells were able to mature their phagosomes into phagolysosomes and displayed the ability to kill internalized bacteria. Importantly, B-1a and B-1b cells effectively present antigen recovered from phagocytosed particles to CD4(+) T cells. However, these cells showed a much lower competence to present soluble antigen or antigen from large, noninternalized particles. B-1 B cells presented particulate and soluble antigen to CD4(+) T cells more efficiently than macrophages, whereas DCs were the most potent APCs. The novel phagocytic and microbicidal abilities identified in B-1 B lymphocytes strengthen the innate nature that has long been attributed to these cells. In the context of adaptive immunity, we show that these innate immune processes are relevant, as they enable B-1 B cells to present phagocytosable particulate antigen. These capacities position these cells at the crossroads that link innate with adaptive immune processes. In a broader context, these newly identified capacities of B-1 B cells further support the previously recognized functional, developmental, and evolutionary relationships between these cells and macrophages.

Pivotal Advance: Peritoneal cavity B-1 B cells have phagocytic and microbicidal capacities and present phagocytosed antigen to CD4+ T cells.

Brechen das lang gehegte Paradigma, dass prim&auml;re B-Zellen nicht phagozytische sind, mehrere Studien k&uuml;rzlich, B-Zellen von Fischen, Amphibien bewiesen haben und Reptilien eine bedeutende phagozytische Kapazit&auml;t haben. Ob dieser Kapazit&auml;t in gewissen S&auml;ugetier-B-Zell-Teilmengen konservierten geblieben ist derzeit ein R&auml;tsel. Hier berichten wir &uuml;ber eine zuvor unbekannte F&auml;higkeit der PerC B-1a und 1 B-b-Lymphozyten, Latex Perlen und Bakterien phagocytose. Im Gegensatz dazu hatte B-2-Lymphozyten eine fast vernachl&auml;ssigbare F&auml;higkeit, diese Partikel zu verinnerlichen. Bei der Phagozytose B-1a und 1 B-b-Zellen konnten ihre Phagosomen in Phagolysosomes &auml;ltere und angezeigt, die F&auml;higkeit, verinnerlichte Bakterien zu t&ouml;ten. B-1a und 1 B-b-Zellen pr&auml;sentieren vor allem effektiv Antigen aus phagozytiertes Teilchen auf CD4(+) T-Zellen wiederhergestellt. Diese Zellen zeigte allerdings eine viel geringere Kompetenz, l&ouml;sliche Antigen oder Antigen aus gro&szlig;en, noninternalized Partikel zu pr&auml;sentieren. B-1 B Zellen pr&auml;sentiert Partikel und l&ouml;sliche Antigen zu CD4(+) T-Zellen effizienter als Makrophagen, w&auml;hrend DCs die potentesten APCs waren. Der Roman phagozytische und Mikrobizide F&auml;higkeiten im B-1 B-Lymphozyten identifiziert st&auml;rken die angeborene Natur, die lange auf diese Zellen zugeschrieben hat. Im Rahmen der adaptiven Immunit&auml;t zeigen wir, dass diese angeborene immun Prozesse relevant, wie sie zu vorhanden phagocytosable Partikel Antigen B-1 B-Zellen aktivieren. Diese Kapazit&auml;ten positionieren diese Zellen an der Kreuzung, die angeborenen mit adaptiven Immunsystems Prozesse verkn&uuml;pfen. In einem breiteren Kontext unterst&uuml;tzen diese neu identifizierten Kapazit&auml;ten von B-1 B-Zellen weiter die zuvor erkannten funktionellen, Entwicklungsst&ouml;rungen und evolution&auml;ren Beziehungen zwischen diesen Zellen und Makrophagen.

Zentrale Vorverkauf: Bauchh&ouml;hle B-1 B-Zellen haben phagozytische und Mikrobizide Kapazit&auml;ten und gegenw&auml;rtigen phagozytiertes Antigen auf CD4 + T-Zellen.

Article	Year
Differentially expressed genes that encode potential markers of goldfish macrophage development in vitro. Developmental and comparative immunology\| PubMed ID: 15043942	2004
Regulation of myeloid development and function by colony stimulating factors. Developmental and comparative immunology\| PubMed ID: 15062647	2004
Development of goldfish macrophages in vitro. Fish & shellfish immunology\| PubMed ID: 15936214	2006
A novel soluble form of the CSF-1 receptor inhibits proliferation of self-renewing macrophages of goldfish (Carassius auratus L.). Developmental and comparative immunology\| PubMed ID: 15978283	2005
A novel hematopoietic granulin induces proliferation of goldfish (Carassius auratus L.) macrophages. The Journal of biological chemistry\| PubMed ID: 16473876	2006
Differential kinase requirements in human and mouse Fc-gamma receptor phagocytosis and endocytosis. Journal of leukocyte biology\| PubMed ID: 16921024	2006
B lymphocytes from early vertebrates have potent phagocytic and microbicidal abilities. Nature immunology\| PubMed ID: 16980980	2006
Cloning and expression analysis of goldfish (Carassius auratus L.) prominin. Fish & shellfish immunology\| PubMed ID: 17123832	2007
Development of macrophages of cyprinid fish. Developmental and comparative immunology\| PubMed ID: 19063916	2009
Microarray analysis of differentially expressed genes from Peyer's patches of cattle orally challenged with bovine spongiform encephalopathy. Journal of toxicology and environmental health. Part A\| PubMed ID: 19697233	2009
Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death. Journal of immunological methods\| PubMed ID: 20381494	2010
Erratum to "Cloning and expression analysis of goldfish (Carasius auratus L.) prominin" [Fish Shellfish Immunol. 22 (2007) 308-317]. Fish & shellfish immunology\| PubMed ID: 20547224	2010
Macrophage activation differentially modulates particle binding, phagocytosis and downstream antimicrobial mechanisms. Developmental and comparative immunology\| PubMed ID: 20600280	2010
Antimicrobial mechanisms of fish leukocytes. Developmental and comparative immunology\| PubMed ID: 21414350	2011
Pivotal Advance: Peritoneal cavity B-1 B cells have phagocytic and microbicidal capacities and present phagocytosed antigen to CD4+ T cells. Journal of leukocyte biology\| PubMed ID: 22058420	2011

Daniel R. Barreda

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